ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial ß-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

Anti-apoptotic MCL-1 promotes long-chain fatty acid oxidation through interaction with ACSL1.

MCL-1 is essential for promoting the survival of many normal cell lineages and confers survival and chemoresistance in cancer. Beyond apoptosis regulation, MCL-1 has been linked to modulating mitochondrial metabolism, but the mechanism(s) by which it does so are unclear. Here, we show in tissues and cells that MCL-1 supports essential steps in long-chain (but not short-chain) fatty acid ß-oxidation (FAO) through its binding to specific long-chain acyl-coenzyme A (CoA) synthetases of the ACSL family. ACSL1 binds to the BH3-binding hydrophobic groove of MCL-1 through a non-conventional BH3-domain. Perturbation of this interaction, via genetic loss of Mcl1, mutagenesis, or use of selective BH3-mimetic MCL-1 inhibitors, represses long-chain FAO in cells and in mouse livers and hearts. Our findings reveal how anti-apoptotic MCL-1 facilitates mitochondrial metabolism and indicate that disruption of this function may be associated with unanticipated cardiac toxicities of MCL-1 inhibitors in clinical trials.

A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.

Functional micropeptides can be concealed within RNAs that appear to be noncoding. We discovered a conserved micropeptide, which we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long noncoding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca(2+) uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca(2+) handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as noncoding.

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3-5. VMATs are also therapeutic drug targets for a number of different conditions6-9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.

A Study of Opiate, Opiate Metabolites and Antihistamines in Urine after Consumption of Cold Syrups by LC-MS/MS.

Studying the origin of opiate and/or opiate metabolites in individual urine specimens after consumption of cold syrups is vital for patients, doctors, and law enforcement. A rapid liquid chromatography-tandem mass spectrometry method using "dilute-and-shoot" analysis without the need for extraction, hydrolysis and/or derivatization has been developed and validated. The approach provides linear ranges of 2.5-1000 ng mL-1 for 6-acetylmorphine, codeine, chlorpheniramine, and carbinoxamine, 2.5-800 ng mL-1 for morphine and morphine-3-ß-d-glucuronide, and 2.5-600 ng mL-1 for morphine-6-ß-d-glucuronide and codeine-6-ß-d-glucuronide, with excellent correlation coefficients (R2 > 0.995) and matrix effects (< 5%). Urine samples collected from the ten participants orally administered cold syrups were analyzed. The results concluded that participants consuming codeine-containing cold syrups did not routinely pass urine tests for opiates, and their morphine-codeine concentration ratios (M/C) were not always < 1. In addition, the distribution map of the clinical total concentration of the sum of morphine and codeine against the antihistamines (chlorpheniramine or carbinoxamine) were plotted for discrimination of people who used cold syrups. The 15 real cases have been studied by using M/C rule, cutoff value, and distribution map, further revealing a potential approach to determine opiate metabolite in urine originating from cold syrups.

A Photoluminescent Colorimetric Probe of Bovine Serum Albumin-Stabilized Gold Nanoclusters for New Psychoactive Substances: Cathinone Drugs in Seized Street Samples.

Screening of illicit drugs for new psychoactive substances-namely cathinone-at crime scenes is in high demand. A dual-emission bovine serum albumin-stabilized gold nanoclusters probe was synthesized and used for quantitation and screening of 4-chloromethcathinone and cathinone analogues in an aqueous solution. The photoluminescent (PL) color of the bovine serum albumin-stabilized Au nanoclusters (BSA-Au NCs) probe solution changed from red to dark blue during the identification of cathinone drugs when excited using a portable ultraviolet light-emitting diodes lamp (365 nm). This probe solution allows the PL color-changing point and limit of detection down to 10.0 and 0.14 mM, respectively, for 4-chloromethcathinone. The phenomenon of PL color-changing of BSA-Au NCs was attributed to its PL band at 650 nm, quenching through an electron transfer mechanism. The probe solution was highly specific to cathinone drugs, over other popular illicit drugs, including heroin, cocaine, ketamine, and methamphetamine. The practicality of this BSA-Au NCs probe was assessed by using it to screen illicit drugs seized by law enforcement officers. All 20 actual cases from street and smuggling samples were validated using this BSA-Au NCs probe solution and then confirmed using gas chromatography-mass spectrometry. The results reveal this BSA-Au NCs probe solution is practical for screening cathinone drugs at crime scenes.

ER-plasma membrane junctions: Why and how do we study them?

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are membrane microdomains important for communication between the ER and the PM. ER-PM junctions were first reported in muscle cells in 1957, but mostly ignored in non-excitable cells due to their scarcity and lack of functional significance. In 2005, the discovery of stromal interaction molecule 1 (STIM1) mediating a universal Ca2+ feedback mechanism at ER-PM junctions in mammalian cells led to a resurgence of research interests toward ER-PM junctions. In the past decade, several major advancements have been made in this emerging topic in cell biology, including the generation of tools for labeling ER-PM junctions and the unraveling of mechanisms underlying regulation and functions of ER-PM junctions. This review summarizes early studies, recently developed tools, and current advances in the characterization and understanding of ER-PM junctions. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

Homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system at ER-PM junctions.

The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-Ca(2+) signaling system is important for cell activation in response to various extracellular stimuli. This signaling system is initiated by receptor-induced hydrolysis of PI(4,5)P2 in the plasma membrane (PM) to generate the soluble second messenger inositol 1,4,5-trisphosphate (IP3). IP3 subsequently triggers the release of Ca(2+) from the endoplasmic reticulum (ER) store to the cytosol to activate Ca(2+)-mediated responses, such as secretion and proliferation. The consumed PM PI(4,5)P2 and ER Ca(2+) must be quickly restored to sustain signaling responses, and to maintain the homeostasis of PI(4,5)P2 and Ca(2+). Since phosphatidylinositol (PI), the precursor lipid for PM PI(4,5)P2, is synthesized in the ER membrane, and a Ca(2+) influx across the PM is required to refill the ER Ca(2+) store, efficient communications between the ER and the PM are critical for the homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system. This review describes the major findings that established the framework of the PI(4,5)P2-Ca(2+) signaling system, and recent discoveries on feedback control mechanisms at ER-PM junctions that sustain the PI(4,5)P2-Ca(2+) signaling system. Particular emphasis is placed on the characterization of ER-PM junctions where efficient communications between the ER and the PM occur, and the activation mechanisms of proteins that dynamically localize to ER-PM junctions to provide the feedback control during PI(4,5)P2-Ca(2+) signaling, including the ER Ca(2+) sensor STIM1, the extended synaptotagmin E-Syt1, and the PI transfer protein Nir2. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions.

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) at the plasma membrane (PM) constitutively controls many cellular functions, and its hydrolysis via receptor stimulation governs cell signaling. The PI transfer protein Nir2 is essential for replenishing PM PIP2 following receptor-induced hydrolysis, but key mechanistic aspects of this process remain elusive. Here, we demonstrate that PI at the membrane of the endoplasmic reticulum (ER) is required for the rapid replenishment of PM PIP2 mediated by Nir2. Nir2 detects PIP2 hydrolysis and translocates to ER-PM junctions via binding to phosphatidic acid. With distinct phosphatidic acid binding abilities and PI transfer protein activities, Nir2 and its homolog Nir3 differentially regulate PIP2 homeostasis in cells during intense receptor stimulation and in the resting state, respectively. Our study reveals that Nir2 and Nir3 work in tandem to achieve different levels of feedback based on the consumption of PM PIP2 and function at ER-PM junctions to mediate nonvesicular lipid transport between the ER and the PM.

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen-Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

Curcumin induces the apoptosis of human monocytic leukemia THP-1 cells via the activation of JNK/ERK pathways.

BACKGROUND: Curcumin is a principal compound of turmeric, commonly used to treat tumors and other diseases. However, its anti-cancer activity in human acute monocytic leukemia THP-1 cells is not clear. This study aimed to study the anti-cancer effect and action of curcumin on THP-1 cells. METHODS: THP-1 parental cells and PMA-treated THP-1 cells, were used as in vitro models to evaluate the anti-cancer effect and mechanism of curcumin. Apoptosis and its mechanism were evaluated by WST-1, flow cytometry and Western blotting. MAPK inhibitors were used to further confirm the molecular mechanism of curcumin-induced THP-1 cell apoptosis. RESULTS: Curcumin induced cell apoptosis of THP-1 cells as shown by cell viability, cell cycle analysis and caspase activity. Curcumin significantly increased the phosphorylation of ERK, JNK and their downstream molecules (c-Jun and Jun B). Inhibitor of JNK and ERK reduced the pro-apoptotic effect of curcumin on THP-1 cells as evidenced by caspase activity and the activation of ERK/JNK/Jun cascades. On the contrary, the pro-apoptotic effect of curcumin was abolished in the differentiated THP-1 cells mediated by PMA. CONCLUSIONS: This study demonstrates that curcumin can induce the THP-1 cell apoptosis through the activation of JNK/ERK/AP1 pathways. Besides, our data suggest its novel use as an anti-tumor agent in acute monocytic leukemia.

Lipid droplets in the nervous system.

Lipid droplets are dynamic intracellular lipid storage organelles that respond to the physiological state of cells. In addition to controlling cell metabolism, they play a protective role for many cellular stressors, including oxidative stress. Despite prior descriptions of lipid droplets appearing in the brain as early as a century ago, only recently has the role of lipid droplets in cells found in the brain begun to be understood. Lipid droplet functions have now been described for cells of the nervous system in the context of development, aging, and an increasing number of neuropathologies. Here, we review the basic mechanisms of lipid droplet formation, turnover, and function and discuss how these mechanisms enable lipid droplets to function in different cell types of the nervous system under healthy and pathological conditions.

Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

S1P(5) is required for sphingosine 1-phosphate-induced autophagy in human prostate cancer PC-3 cells.

Sphingosine 1-phosphate (S1P) is a platelet- and endothelial cell-released lysophospholipid that regulates various cellular functions through activating a specific family of G protein-coupled receptors. Both platelet activation and angiogenesis play important roles in cancer development, implying that cancer cells might encounter a large amount of S1P during these processes. Cancer cells, in the meantime, may experience nutrient deprivation and rely on autophagy for early development. Whether extracellular S1P regulates autophagy remains to be tested. In the present work, we investigated whether autophagy is regulated by S1P in PC-3 cells. Through monitoring the modification patterns of LC3 by Western blotting, we demonstrated that autophagy was induced by exogenously applied S1P in PC-3 cells. This observation was further confirmed by fluorescence microscopy using PC-3 cells stably expressing enhanced green fluorescent protein-LC3. By applying small interfering RNA and dihydro-S1P, S1P(5) activation was found to be involved in this process. Besides, mammalian target of rapamycin signaling was inhibited upon S1P treatment. Taken together, our results suggest that, under serum-starved conditions, S1P further upregulates autophagic activity through S1P(5)-dependent pathways in PC-3 cells.

Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells.

Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM) important for Ca2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM junctions, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P2 replenishment following receptor stimulation, as a readout of PI transfer, using a PI(4,5)P2 biosensor and total internal reflection fluorescence microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM junctions and mediate PI(4,5)P2 replenishment with PI in the ER in stimulated cells. These methods provide spatial and temporal analysis of ER-to-PM PI transfer during receptor stimulation and can be adapted to other research questions related to this topic.

Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin's dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

Lysophosphatidic acid-induced interleukin-1 beta expression is mediated through Gi/Rho and the generation of reactive oxygen species in macrophages.

Lysophosphatidic acid (LPA), a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoproteins, is known to regulate inflammation and atherosclerosis by binding to its cognate receptors. In this study, we reported that LPA upregulated interleukin-1 beta (IL-1 beta) expression in mouse J774A.1 macrophages. By using pharmacological inhibitors, it was suggested that G(i)/Rho activation and subsequent reactive oxygen species (ROS) production were involved in IL-1 beta induction. In addition, IL-1 beta induction by LPA was also observed in human primary macrophages. In summary, LPA is involved in the processes of inflammation by affecting macrophage behavior.

Lysophosphatidic acid-induced oxidized low-density lipoprotein uptake is class A scavenger receptor-dependent in macrophages.

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoprotein (OxLDL). It is suggested that LPA is involved in atherosclerosis, and our previous studies showed that LPA regulates inflammation in multiple cell types. The main aim of this study was to investigate the effects of LPA on the uptake of OxLDL by mouse J774A.1 macrophages. We observed that LPA upregulated fluorescence-labeled DiI-OxLDL uptake in J774A.1 cells. Meanwhile, expression of the class A scavenger receptor (SR-A), a receptor for modified LDL, was also enhanced. Furthermore, pertussis toxin (PTx) or Ki16425 significantly abolished LPA's effects, indicating that G(i) and LPA(3) are involved in OxLDL uptake and SR-A expression. Of most importance, the LPA-induced OxLDL uptake could be inhibited when cells were incubated with a functional blocking antibody of SR-A. Our results suggest that LPA-enhanced OxLDL uptake is mediated via LPA(3)-G(i) activation and subsequent SR-A expression.